Long-term storage of non-glycosylated recombinant human G-CSF

ABSTRACT

The present invention provides a method for stable long-term storage of non-glycosylated recombinant human Granulocyte-Colony Stimulating Factor (“G-CSF”), wherein an aqueous acetate or glutamate buffered G-CSF composition containing the non-glycosylated recombinant human G-CSF and sorbital is cooled to a temperature of −15° C. or below to obtain a frozen G-CSF composition, which frozen composition is then stored in the frozen state and then increased in temperature to a temperature within the range of from 2° C. to 8° C. for a period of time adjusted to allow the composition to thaw and to obtain a liquid composition having a G-CSF content of at least 95% of the G-CSF content of the original composition.

This application is a national phase entry of PCT Internationalapplication number PCT/EP2011/060454, filed Jun. 22, 2011. Thisapplications also claims the benefit of the earlier filing dates ofEuropean application EP 10166915.8, filed Jun. 22, 2010, and USprovisional application 61/360,562, filed Jul. 1, 2010.

FIELD OF THE INVENTION

The present invention relates to a method for long-term storage ofnon-glycosylated recombinant human G-CSF.

BACKGROUND OF THE INVENTION

G-CSF (Granulocyte-Colony Stimulating Factor) is a naturally occurringgrowth factor which belongs to the family of cytokines. G-CSF plays acrucial role in hematopoesis and enhances maturation, proliferation,differentiation and survival of neutrophils and neutrophilic successorcells. Clinically, G-CSF is mainly used for controlling tumors and, inparticular, for the treatment of neutropenia following chemotherapy, andit is also applied for bone marrow transplantations and in the treatmentof infectious diseases.

Human G-CSF in its naturally occurring form is an about 20 kDaglycoprotein which has five cysteine residues. Four of these residuesform two intramolecular disulfide bridges which are crucial for theactivity of the protein. As G-CSF is obtained only in small amounts fromnatural sources, mainly recombinant forms of G-CSF are used inmedicaments, in particular those which have been produced by expressingthe protein in prokaryotic hosts. Proteins expressed in prokaryotichosts such as E. coli differ from natural occurring G-CSF in that theyare not glycosylated. Proteins expressed in E. coli have an additionalN-terminal methionine residue necessary for expression in this hostorganism.

Due to the high hydrophobicity of the protein, non-glycosylatedrecombinant G-CSF is relatively unstable. The molecule easily adsorbs tothe inner surface of storage vessels, vials, syringes or the like andforms dimers and higher aggregates. Conventional liquid G-CSFformulations also are sensitive to mechanical stress, for example as aresult of shaking during transport, and to accidental freezing andthawing, which may also result in higher levels of aggregates and lossof biological activity. Moreover, G-CSF is subject to chemicalmodifications such as deamidation, oxidation, cleavage of disulfidebridges or proteolysis. Deamidation, which occurs more rapidly thanother degradation routes, is a particular problem due to the highglutamine content of G-CSF. Altogether, this may result in a reducedcontent of biologically available and active monomeric G-CSF,particularly upon prolonged storage of the protein. This is not onlycostly but also is undesirable for therapeutic reasons, for example ifthe G-CSF is to be administered over a prolonged period of time at aconstant dosage. Furthermore, products formed by multimerization ordeamidation may result in an undesired immune response.

Stabilization of G-CSF formulations is subject of various patent andnon-patent literature.

DE-A-37 23 781 describes aqueous phosphate-buffered G-CSF formulationscontaining pharmaceutically acceptable surfactants such aspolyoxyethylene sorbitan esters which are used in combination with humanserum albumin and mannitol for stabilizing the active ingredient. Theseformulations are stable at 4° C. over a prolonged period of time. Due totheir antigenic properties, however, proteins and peptides of human andanimal origin may cause undesired immunological reactions.

EP-A-0 373 679 discloses G-CSF formulations having a pH value of from2.75 to 4.0 and low conductivity, which may be stored over prolongedperiods of time without formation of aggregates. If any, buffer is usedin these formulations in small amounts of less than 2 mM in order toavoid the aggregation of G-CSF.

EP-A-1 197 221 discloses long-term stable G-CSF formulations at a pH offrom 5 to 7, which contain one or more amino acids of the group oflysine, histidine, arginine, aspartic acid, glutamic acid, threonine andasparagine, as well as one or more hydrophobic amino acids. Methionineis added to prevent oxidation of methionine residues in the G-CSFmolecule.

WO-A-2007/034509 discloses stable aqueous formulations containingrecombinant human G-CSF and an amino acid which is an oxidationsuppressant for the methionine residues in the protein.

WO-A-2005/042024 discloses pharmaceutical compositions comprising G-CSFand an acid such as acetic acid or glutamic acid, which is free ofsurfactants.

WO-A-2005/039620 discloses succinate- and tartrate-buffered compositionsstable over a wide pH range.

Herman, A. C. et al. (“Characterisation, Formulation, and Stability ofNeupogen® (Filgrastim), a Recombinant Human Granulocyte-ColonyStimulating Factor.” In: Formulation Characterisation and Stability ofProtein Drugs, pp. 303-328, R. Pearlman and Y. J. Wang, Eds., PlenumPress, New York, 1996) describe stabilized compositions ofnon-glycosylated recombinant G-CSF which contain 10 mM of sodiumacetate, pH 4.0, 5% of mannitol and 0.004% of Polysorbate 80. Suchcompositions are stable for more than 24 months at 2-8° C. Substitutingmannitol with sorbitol in a filgrastim formulation was found toeliminate sensitivity of the protein to aggregation during inadvertentfreezing and thawing. Storing in a freezer, however, is to be avoidedaccording to the manufacturer's instructions.

WO-A-2007/099145 discloses liquid acetate-buffered G-CSF formulationscomprising polysorbate 20 and/or polysorbate 80 as a surfactant andhaving a pH-value between 4.1 and 4.4

WO-A-2008/122415 discloses liquid aqueous glutamate-buffered G-CSFformulations having a pH of from 3.5 to 4.8 which are stable underconditions of mechanical stress encountered, for example, upon freezingand thawing.

OBJECTS AND SUMMARY OF THE INVENTION

Piedmonte et al. (Pharmaceutical Research, Vol. 24, No. 1, January 2007,pp: 136-146) describe the effect of sorbitol on protein aggregation infrozen protein formulations.

The object of the present invention was to provide a method for stablelong-term storage of biologically active non-glycosylated recombinanthuman G-CSF, wherein degradation, in particular deamidation, and loss ofG-CSF during storage due to adsorption phenomena to container walls isreduced.

This object is achieved by the method of the present invention forstable long-term storage of non-glycosylated recombinant human G-CSF,said method comprising the steps of:

-   -   (a) providing an aqueous acetate or glutamate buffered G-CSF        composition containing the non-glycosylated recombinant human        G-CSF and sorbitol;    -   (b) cooling the G-CSF composition provided in step (a) to a        temperature of −15° C. or below to obtain a frozen G-CSF        composition;    -   (c) storing the G-CSF composition obtained in step (b) in the        frozen state; and    -   (d) increasing the temperature of the frozen G-CSF composition        of step (c) to a temperature within the range of from 2° C. to        8° C. over a period of time adjusted to allow the composition to        thaw and to obtain a liquid composition having a G-CSF content        of at least 95% of the G-CSF content of the composition provided        in step (a).

The present invention further relates to a method of providing apharmaceutical composition of non-glycosylated recombinant human G-CSF,said method comprising the steps of:

-   -   (a) formulating the non-glycosylated recombinant human G-CSF        with an acetate or glutamate buffer and sorbitol to obtain an        aqueous buffered G-CSF composition;    -   (b) cooling the G-CSF composition of step (a) to a temperature        of −15° C. or below to obtain a frozen G-CSF composition;    -   (c) storing the G-CSF composition obtained in step (b) in the        frozen state;    -   (d) increasing the temperature of the frozen G-CSF composition        of step (c) to a temperature within the range of from 2° C. to        8° C. over a period of time adjusted to allow the composition to        thaw and to obtain a liquid composition having a G-CSF content        of at least 95% of the G-CSF content of the composition provided        in step (a); and    -   (e) filling the liquid composition obtained in step (d) into        primary packagings for parenteral use.

In the course of the invention it has been found that by the method ofthe invention deamidation and loss of non-glycosylated recombinant humanG-CSF can considerably be reduced or even avoided, even if the G-CSF isprovided in high concentrations and in large volumes and without the useof surfactants. In this way, activity is maintained even at prolongedstorage. Moreover, as G-CSF compositions can be stored in the frozenstate, they are not sensitive to mechanical stress as may beexperienced, for example, during transport.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows a comparison of deamidation products observed withfilgrastim samples subjected to the method of the invention (lanes 1 and2) and filgrastim samples stored at a temperature of 25° C. (lanes 3 and4) as determined by Isoelectric Focussing (IEF).

DETAILED DESCRIPTION OF THE INVENTION

The non-glycosylated recombinant human G-CSF protein used in thecompositions of the present invention (in the following also referred toas G-CSF) may be any protein comprising the non-glycosylated amino acidsequence of human G-CSF and having the biological activity thereof.Non-glycosylated recombinant human G-CSF is typically obtained byexpressing the human G-CSF gene in a prokaryotic host such as E. coli.Non-glycosylated recombinant human G-CSF expressed in E. coli typicallyhas an N-terminal Met residue. In a preferred embodiment of theinvention, the human G-CSF comprises or has the primary structure ofhuman G-CSF plus an N-terminal methionine (r-met HU G-CSF) as indicatedin the European Pharmacopoeia 6.3 Monograph (01/2009:2206; “FilgrastimConcentrated Solution” page 4142) or in Herman, A. C. et al. (supra),i.e., the amino acid sequence of filgrastim, or is a variant thereofhaving essentially the biological activity of filgrastim, for example avariant having N-terminal or C-terminal extensions such as fusionproteins, a variant wherein the methionine residue at the N-terminal endhas been replaced by some other amino acid such as glycine, or a varianthaving neutral mutations in the amino acid sequence. G-CSF variantsuseful in the formulations of the present invention are disclosed, e.g.,in EP-A-0 456 200.

The buffer system used in the G-CSF compositions of the presentinvention is an acetic acid/acetate buffer or a glutamic acid/glutamatebuffer. The composition used in the invention is preferably free ofother buffering agents. The buffers used according to the invention canbe prepared, for example, starting from acetic acid or glutamic acidand/or a salt thereof and adjusting the pH to the desired value usingthe corresponding acid or base or another suitable inorganic or organicacid or inorganic base such as hydrochloric acid or an alkali hydroxideor alkaline earth hydroxide. Physiologically acceptable acetic acidsalts or glutamic acid salts are preferred, e.g., alkali, alkalineearth, or ammonium salts. Alkali or ammonium salts are preferred, inparticular the monosodium salt. Preferably, the buffer is preparedstarting from acetic acid or glutamic acid and the pH value is adjustedusing a suitable inorganic base, for example sodium hydroxide.

The pH value of the composition provided in step (a) of the process ofthe invention is typically in the range of from 3.5 to 5.0, preferablyin the range of from 3.7 to 4.8. More preferably the pH is in the rangeof from 3.7 to 4.6, for example of from 4.0 to 4.6.

The concentration of the acetate or glutamate buffer is advantageouslyadjusted so as to achieve a pH-stabilizing effect at the desired pHvalue and a sufficient buffer capacity. Usually, the acetate orglutamate buffer has a concentration of at least 0.5 mM, preferably offrom 1 to 100 mM, and more preferably of from 2 to 80 mM. Bufferconcentrations in the range of from 2 to 40 mM, in particular of from 2to 25 mM, for example of from 5 to 15 mM and preferably about 10 mM,will provide sufficient stability and will be low enough to avoidundesired tissue reactions upon injection of the composition.

The G-CSF concentration in the composition provided in step (a) of themethod of the invention will depend on the intended use. The upperconcentration limit results from the solubility of G-CSF in the buffer.Typically, the G-CSF concentration is in a range of from 0.1 to 8 mg/ml,preferably of from 0.25 to 6.5 mg/ml. In analytical samples or inpharmaceutical compositions to be administered without further dilution,G-CSF is present in an amount which typically is in a range of from 0.1to 2.0 mg/ml, preferably up to 2.5 mg/ml. In more concentratedcompositions, for example compositions containing G-CSF as a processintermediate, which may be further processed to obtain the drug productsuitable for administration to a patient, the G-CSF concentrationtypically is in a range of from 2.5 mg/ml to 8.0 mg/ml, preferably offrom 2.5 to 6.5 mg/ml, and more preferably up to 5.5 mg/ml.

The composition provided in step (a) of the method of the inventioncomprises sorbitol as a tonicity modifier. Preferably, sorbitol is theonly tonicity modifier used in the composition except for the buffersystem. Sorbitol is typically present in an amount of up to 200 mg/ml,preferably of from 10 to 100 mg/ml, more preferably of from 25 to 75mg/ml, for example about 50 mg/ml.

The compositions used in the method of the invention may or may notcomprise a surfactant. If a surfactant is present, the surfactanttypically is a non-ionic surfactant. Preferably, the non-ionicsurfactant is selected from the group consisting of fatty alcoholethoxylates, alkylpolyglycosides, polyoxyalkylenes, polysorbates ormixtures of two or more thereof. Polyoxyalkylenes such aspolyoxyalkylene block copolymers, for example Poloxamer 188 (availableunder the trade name PLURONIC F68), and polysorbates, i.e.,polyoxyethylene sorbitan esters of aliphatic fatty acids are preferred.Most preferred are polysorbates such as polyoxyethylene sorbitanmonolaurate (available under the trade name TWEEN 20), polyoxyethylenesorbitan monopalmitate (TWEEN 40), polyoxyethylene sorbitan monostearate(TWEEN 60), polyoxyethylene sorbitan tristearate (TWEEN 65),polyoxyethylene-sorbitan monooleate (TWEEN 80) and polyoxyethylenesorbitan trioleate (TWEEN 85). Polyoxyethylene sorbitan monolaurate andpolyoxyethylene sorbitan monooleate are most preferred.

If a surfactant is used, the surfactant is preferably present in anamount of 5 mg/ml or less, preferably 1 mg/ml or less. Preferably,surfactants, in particular polysorbates, are used in amounts of from0.001 to 1.0 mg/ml, more preferably of from 0.01 to 0.5 mg/ml.

While the composition provided in step (a) of the method of theinvention may comprise further agents such as amino acids, reducingagents, antioxidants and serum proteins, the composition typicallyconsists of G-CSF, the aqueous acetate or glutamate buffer, sorbitoland, optionally, a surfactant, and thus is free of other agents.

The compositions provided in step (a) of the method of the invention maybe prepared in a manner known per se. For example, the buffersubstances, i.e., acetic acid or glutamic acid or a salt thereof, thesorbitol and, optionally, other additives such as surfactants aredissolved in a suitable amount of an aqueous solvent, usually water. Ifnecessary, the pH value is adjusted using a suitable acid or base asdescribed above. Following sterilization, for example by filtrationthrough a sterile filter, G-CSF is added in the desired concentration.Alternatively and preferably, the G-CSF composition used in step (a) isobtained as a batch from the production process with or withoutre-buffering.

The aqueous G-CSF composition provided in step (a) of the method of theinvention can be provided in any desired volume but preferably has avolume in the range of from 0.1 ml to 8 l, preferably of from 5 ml to 4l, more preferably of from 10 ml to 2.0 l, and most preferably of from100 ml to 1.5 l. The composition is provided in a suitable containersuch as a polyethylene (PE) bag, a glass bottle, or a bottle made ofpolyethylene terephthalate (PET) without or with glycol (PETG).

Filling the composition into the container is typically carried outunder sterile conditions and preferably using an inert gas such asnitrogen. Typically, containers are filled only partially with thecomposition and preferably up to a volume of not more than 90%, theheadspace in the containers being preferably filled with the inert gas.

The liquid aqueous G-CSF composition of the present invention providedin the desired volume is cooled down to a temperature of −15° C. orbelow until frozen. Typically, the compositions are cooled to atemperature of between −15 and −25° C., for example about −20° C., orthey are cooled to a temperature of between −60° C. and −80° C. Coolingcan be effected, for example, in a freezer or a cold room or bysubmerging the containers with the G-CSF composition into liquidnitrogen.

The frozen G-CSF composition obtained in step (b) is stored in thefrozen state at the desired temperature of −15° C. or below. Typically,the composition is stored at the temperature to which the compositionhas been cooled, i.e., as described above, preferably at a temperatureof between −15 and −25° C. or between −60 and −80° C., which is thetemperature standard cold rooms or deep freezers are defined to.Typically, the frozen G-CSF composition is stored over a period of atleast two days, preferably at least one month, for example for at leastthree months or at least six months. It has been found that deamidationis considerably reduced during the time period where the G-CSFcomposition is stored in the frozen state (see Example 4 and FIG. 1).

Following storing of the frozen G-CSF composition in step (c), thetemperature of the frozen composition is increased to a temperaturewithin the range of from 2° C. to 8° C. over a period of time adjustedto allow the composition to thaw and to obtain a liquid compositionhaving a G-CSF content of at least 95% of the G-CSF content of thecomposition provided in step (a).

The term “increasing the temperature to a temperature within the rangeof from 2° C. to 8° C.” means that the composition is not exposed to atemperature above 8° C. Specifically, according to one embodiment of theinvention, the frozen compositions can be warmed to a temperaturebetween 2° C. and 8° C. by gradually or linearly increasing thetemperature over an extended period of time. The extended period of timenecessary to thaw the frozen composition and to obtain a liquidcomposition having a G-CSF content which is at least 95% of the G-CSFcontent of the composition originally provided is typically at least 6hs. For example, in case a composition is kept in the frozen state at atemperature of −20° C., the composition can be warmed from −20° C. to+4° C. over a period of 6 hs with a gradual or linear hourly temperatureincrease of 4° C. A linear temperature gradient can be run, for example,using the Integrated Biosystems CryoPilot™ System.

According to a preferred embodiment of the invention, the frozencomposition will be immediately transferred to the desired temperaturebetween 2° C. and 8° C. and will then be maintained at that temperaturefor an extended period of time to allow the frozen composition to thawand to obtain a liquid composition having the required G-CSF content.Typically, the frozen composition is transferred to a cold room or awater bath adjusted to this temperature range. In this case, the periodof time for which the composition is maintained at that temperaturedepends, e.g., on the volume of the frozen G-CSF composition and theconcentration of G-CSF in the composition. As a rule, the period of timerequired for a composition having a large volume, e.g., 100 ml or more,and a high G-CSF concentration is longer than for a composition having asmall volume, e.g., below 100 ml, and a low G-CSF concentration.Likewise, the period of time required in a cold room is longer than in awater bath. Generally, the time period required to obtain the desiredhigh G-CSF content is at least 12 hours and typically the time period isin the range of from 12 to 72 hours. For example, the time required fora composition having a small volume of below 100 ml generally is in therange of from 12 to 24 hours, while the time required for a compositionhaving a large volume of 100 ml or more, for example of from 100 ml to 8l, such as 0.8 l, and/or a high concentration of G-CSF, for example offrom 2.5 to 8 mg/ml, is typically 18 hours or more, for example 24 to 48hours in a water bath and 36 hours or more, for example 36 to 72 hours,in a cold room. Still larger volumes may require proportionally longertimes.

In the liquid compositions obtained, the G-CSF content is at least 95%,preferably at least 97%, and most preferably at least 99%, of the G-CSFcontent of the composition provided in step (a). The term“G-CSF-content” is meant to encompass monomeric G-CSF and multimersthereof as well as related proteins derived therefrom such as deamidatedand oxidized variants. The G-CSF-content can be determined, for example,by Size Exclusion Chromatography (SEC) or by Reversed Phase-HPLC(RP-HPLC) as described in the European Pharmacopoeia 6.3 Monograph(01/2009:2206; “Filgrastim Concentrated Solution” pages 4143-4144, inparticular page 4143: “Impurities with molecular masses higher than thatof filgrastim. Size-exclusion chromatography (2.2.30)” and “Relatedproteins. Liquid chromatography (2.2.29)”). While both methods give thesame results, typically RP-HPLC is used.

Using the method of the invention, the biological potency of therecombinant G-CSF obtained in step (d) is essentially maintained.Specifically, the biological potency is at least 90%, preferably atleast 95%, and more preferably at least 97%, 98% or 99% relative to thebiological potency of the G-CSF provided in step (a). Biologicalactivity is determined as described for filgrastim in the EuropeanPharmacopoeia 6.3 Monograph (01/2009:2206; “Filgrastim ConcentratedSolution”; pages 4142-4144, in particular page 4144: ASSAY—“Potency”).In brief, biological potency of the composition obtained in step (d) isdetermined by measuring its ability to stimulate proliferation of NFS-60cells compared with the composition provided in step (a) calibrated inInternational Units as a reference. To determine the number of viablecells, intracellular ATP may be quantified using a luciferasechemiluminescence system. The measured luminescent signal isproportional to the amount of ATP which is directly proportional to thenumber of cells present. Relative potency may be calculated using asuitable statistical method, for example the parallel line assayaccording to European Pharmacopoeia 5.3 Monograph, and is expressed inpercent of the composition obtained in step (d) compared to thecomposition of step (a).

Following step (d), in particular in the method of providing apharmaceutical composition of non-glycosylated recombinant human G-CSF,the obtained liquid composition may be filled into primary packagingsfor parenteral use such as vials or syringes. Advantageously, the liquidcomposition may be divided into aliquots suitable for administration toa patient, for example, by injection or infusion, before filling.Concentrated solutions of G-CSF may be diluted before filling, and,optionally, the dilution buffer may also contain surfactant and otheradditives.

The G-CSF formulations obtained after storing and thawing in steps (c)and (d) of the method of the invention show no or only a minor loss ofG-CSF protein due to adsorption, deamidation or aggregation of theprotein. As described above, the G-CSF composition finally obtainedaccording to the method of the invention has an overall G-CSF content ofat least 95% of the initial content of G-CSF. These compositions,optionally after dilution, may be used as pharmaceuticals in variousapplication forms, for example preparations for injection or infusion,in particular for intravenous, intramuscular, or subcutaneousadministration. The pharmaceuticals obtained may be used for anyindication for which G-CSF may be employed, such as for the treatment ofneutropenia, for bone marrow transplantations, and in the treatment ofinfectious diseases and of tumor diseases.

The present invention will now be illustrated in more detail withreference to the following examples and to FIG. 1, which are notintended to limit the invention.

EXAMPLES Methods

1. Size Exclusion Chromatography (SEC)

Aggregation analysis by SEC was performed according to the methoddescribed in the European Pharmacopoeia 6.3 Monograph (01/2009:2206;“Filgrastim Concentrated Solution” page 4143: “Impurities with molecularmasses higher than that of filgrastim. Size-exclusion chromatography(2.2.30)”) except that fluorescence detection was used. Briefly,hydrophilic silica gel was used as a stationary phase at a temperatureof 30° C. Elution was carried out using a phosphate buffered ammoniumhydrogen carbonate solution as a mobile phase at a flow rate of 0.5ml/min. Fluorescence detection was at 345 nm and excitation was at 280nm. The chromatograms were quantified, differentiating G-CSF monomersfrom higher aggregates as impurities. Results of experiments areexpressed as percent peak area (%).

2. Reversed Phase (RP) HPLC

G-CSF content and impurities (deamidated and oxidized variants) insamples after long term storage using RP-HPLC were determined accordingto the method described in the European Pharmacopoeia 6.3 Monograph(01/2009:2206; “Filgrastim Concentrated Solution”, page 4143: “Relatedproteins. Liquid chromatography (2.2.29)”) except that fluorescencedetection was used for determination of purity as described above.Protein content was determined against a G-CSF reference standard by UVdetection at 215 nm. Results of experiments are expressed as percentpeak area (%).

3. Isoelectric Focussing (IEF)

Analysis of samples after freezing and thawing for impurities withcharges differing from that of filgrastim was carried out by IEFaccording to the method described in the European Pharmacopoeia 6.3Monograph (01/2009:2206; “Filgrastim Concentrated Solution”, page 4143:“Impurities with charges differing from that of filgrastim. Isoelectricfocusing (2.2.54)”) except that reference solutions with a lowerconcentration and silver staining were used to achieve highersensitivity.

Example 1

The following aqueous acetate or glutamate buffered compositions offilgrastim as a non-glycosylated recombinant human G-CSF were preparedas shown in Table 1 below and used in the following experiments:

TABLE 1 Composition G-CSF (mg/ml) Buffer (10 mM) Sorbitol pH 1 2.26Acetate 50 mM 4.1 2 1.90 Glutamate 50 mM 4.4 3 3.10 Acetate 50 mM 4.5 43.30 Acetate 50 mM 4.5 5 1.98 Glutamate 50 mM 4.4 6 1.86 Glutamate 50 mM4.4 7 1.70 Glutamate 50 mM 4.4 8 1.79 Glutamate 50 mM 4.4

The G-CSF content in mg/ml of each composition was defined to be the100% value in all following experiments.

Example 2

Compositions 1 and 2 (30 ml in a polyethylene (PE) bag) were subjectedto freezing and thawing under various conditions using the IntegratedBiosystems CryoPilot™ System. Percentages of aggregates, oligomers,dimers and monomers of G-CSF as well as overall G-CSF content weredetermined using SEC as described in the European Pharmacopoeia 6.3Monograph (01/2009:2206; “Filgrastim Concentrated Solution”, page 4143:“Impurities with molecular masses higher than that of filgrastim.Size-exclusion chromatography (2.2.30)”). All values were determinedbefore freezing (T0) and after freezing and thawing following conditionsA, B and C. Results and Freeze/Thaw (F/T) conditions are indicated inTable 2 below.

TABLE 2 F/T Olig- G-CSF Composi- condi- Aggregate omers Dimers Monomerscontent tion tions (%) (%) (%) (%) (%) 1 T0 <0.1 <0.1 0.3 99.7 100.2 A<0.1 <0.1 0.4 99.6 85.0 B <0.1 <0.1 0.4 99.5 98.7 C <0.1 <0.1 0.4 99.5102.9 2 T0 <0.1 <0.1 0.2 99.8 100.0 A <0.1 <0.1 0.3 99.7 74.3 B <0.1<0.1 0.4 99.6 100.6 C <0.1 <0.1 0.4 99.6 100.8F/T Conditions:

T0: Compositions before freezing

A: Compositions were quickly cooled to −20° C. Following storage of thecompositions at this temperature for 4 hs, the frozen compositions wereimmediately transferred to a temperature of +20° C. Compositions werekept for 2 hs at that temperature and then were transferred to atemperature of +4° C. where they were kept for further 12 hs.Compositions had completely thawed after 2 hs at +20° C. and 1 h at 4°C.

B: Compositions were quickly cooled from +4° C. to −20° C. Followingstorage for 4 hs at that temperature, temperature was slowly increasedto a temperature of +4° C. over a period of 7 hs using the programmedCryoPilot™ linear temperature gradient.

C: Compositions were cooled from +4° C. to −20° C. over a period of 17hs using the programmed CryoPilot™ linear temperature gradient.Thereafter, temperature was slowly raised to a temperature of +4° C.over a period of 7 hs using a programmed temperature gradient of theCryoPilot™ system.

The results show that quick thawing under condition A results in asignificant loss of G-CSF content, while slowly increasing thetemperature of the frozen compositions as under conditions B and C from−20° C. to +4° C. allows to obtain liquid compositions having a G-CSFcontent comparable to the G-CSF content of the originally provided G-CSFcomposition. Freezing rate has no effect on the final G-CSF content.

Example 3

Compositions 3 and 4 (3.5 ml in 5 ml PETG bottles) were subjected to 5consecutive F/T cycles. In each cycle, samples were cooled from +4° C.to −20° C. in a freezer and after storage for 20 hours at −20° C. weredirectly transferred to a cold room adjusted to a temperature of +4° C.Samples were left at that temperature for a period of 16 hs for thawingand allowing the compositions to regain their original content in G-CSF.G-CSF content and impurities were determined at the beginning of theexperiment and after cycle 1, 3 and 5 using SEC (multimers offilgrastim) and RP-HPLC (G-CSF content and deamidated and oxidized G-CSFvariants) as described above. The results are shown in Table 3 below.

TABLE 3 RP-HPLC SEC G-CSF G-CSF Sum Sum Content Content impuritiesimpurities Composition F/T cycle (%) (mg/ml) (%) (%) 3 0 100 3.1 1.5 1.51 100 3.1 1.5 1.7 3 100 3.1 1.5 1.8 5 100 3.1 1.6 1.8 4 0 100 3.3 1.22.5 1 103 3.4 1.3 2.6 3 103 3.4 1.5 2.7 5 103 3.4 1.5 2.7

As may be seen from the above results, the G-CSF content as well as thesum of impurities remain essentially the same after each cycle, allvalues being within the limits of experimental error.

Example 4

Compositions 5 and 6 (7 ml in 10 ml PETG bottles) were cooled from +4°C. to −20° C. in a freezer. Frozen samples were stored for two months at−20° C. and then directly transferred to a cold room adjusted to atemperature of +4° C. Samples were left at that temperature for a periodof 24 hs for thawing and allowing the compositions to regain theiroriginal content in G-CSF. For the time of the experiment, samples ofcompositions 5 and 6 were kept at 25° C. as a control.

All samples were analyzed for impurities with charges differing fromthat of filgrastim using Isoelectric Focussing (IEF) as described above.The results are shown in FIG. 1, wherein the principal band, i.e., themost intense band, represents filgrastim, and bands having lowerintensities migrating below the main band represent mainly deamidatedvariants thereof. As will be seen from FIG. 1, samples stored at −20° C.(lanes 1 and 2) show considerably less deamidated variants of filgrastimthan samples stored at 25° C. (lanes 3 and 4).

Furthermore, overall G-CSF content and impurities of the samples storedat −20° C. were determined using RP-HPLC (G-CSF content and deamidatedand oxidized G-CSF variants) and SEC (multimers of G-CSF) as describedabove before freezing (T0) and following storage for two months andthawing (T1/−20° C.). The results are shown in Table 4 below togetherwith the results for the control samples stored at 25° C. (T1/+25° C.).

TABLE 4 RP-HPLC SEC G-CSF G-CSF Sum Sum Time of Content Contentimpurities impurities Composition Testing (%) (mg/ml) (%) (%) 5 T0 1001.98 1.5 0.2 T1/−20° C. 100 1.98 1.5 0.4 T1/+25° C. 100 1.98 2.9 0.1 6T0 100 1.86 2.6 0.2 T1/−20° C. 99 1.85 2.1 0.4 T1/+25° C. 99 1.84 3.60.1

As may be seen from the above results, the G-CSF content remainsessentially the same before and after freezing, all values being withinthe limits of experimental error. The increase in deamidated andoxidized G-CSF variants as determined by RP-HPLC is in conformity withthe results obtained using IEF shown in FIG. 1.

Example 5

Compositions 5, 7 and 8 (800 ml in 1000 ml PETG bottles) were stored for36 months at −20° C. Following storage, the compositions weretransferred to a cold room adjusted to a temperature of +4° C. and leftat that temperature for thawing for a period of 48 hours. The liquidcompositions thus obtained were subjected to 5 consecutive F/T cycles.In each cycle, samples were cooled from +4° C. to −20° C. in a freezerand after storing for at least 24 hours at −20° C. were directlytransferred to a cold room adjusted to a temperature of +4° C. Sampleswere left at that temperature for a period of at least 48 hs for thawingand allowing the compositions to regain their original content in G-CSF.G-CSF content and impurities were determined at the beginning of theexperiment (F/T0) and after completion of cycle 5 (F/T5) using RP-HPLC(G-CSF content and deamidated and oxidized G-CSF variants) and SEC(multimers of G-CSF) as described above. The results are shown in Table5 below.

TABLE 5 RP-HPLC SEC G-CSF G-CSF Sum Sum Content Content impuritiesimpurities Composition F/T cycle (%) (mg/ml) (%) (%) 5 0 100 1.98 1.50.3 5 99 1.96 1.2 0.3 7 0 100 1.7 2.7 0.3 5 102 1.73 2.5 0.3 8 0 1001.79 2.6 0.3 5 102 1.82 2.3 0.3

As may be seen from the above results, the G-CSF content as well as thesum of impurities before freezing and after completion of cycle 5 remainessentially the same, all values being within the limits of experimentalerror.

The invention claimed is:
 1. A method for stable long-term storage ofnon-glycosylated recombinant human Granulocyte-Colony Stimulating Factor(G-CSF), said method comprising the steps of: (a) providing an aqueousacetate or glutamate buffered G-CSF composition containing anon-glycosylated recombinant human G-CSF and sorbitol, wherein theamount of G-CSF is in the range of from 0.1 mg/ml to 8.0 mg/ml andwherein said composition has a volume of from 100 ml to 8 l; (b) coolingthe G-CSF-formulation provided in step (a) to a temperature of −15° C.or below to obtain a frozen G-CSF formulation; (c) storing theG-CSF-formulation obtained in step (b) in the frozen state over a periodof at least one month; (d) increasing the temperature of the frozenG-CSF composition of step (c) to a temperature within the range of from2° C. to 8° C. over a period of time of at least 6 hours to allow thecomposition to thaw and to obtain a liquid composition having a G-CSFcontent of at least 95% of the G-CSF content of the composition providedin step (a).
 2. The method of claim 1, wherein the amount of G-CSF inthe buffered G-CSF formulation of step (a) is in the range of from 0.25mg/ml to 6.5 mg/ml.
 3. The method of claim 1, wherein the buffered G-CSFcomposition has a pH of from 3.5 to
 5. 4. The method of claim 1, whereinthe acetate or glutamate concentration of the buffered G-CSF compositionis in the range of from 0.5 mM to 100 mM.
 5. The method of claim 1,wherein the amount of sorbitol in the G-CSF composition is in the rangeof from 10 to 100 mg/ml.
 6. The method of claim 1, wherein the aqueousacetate or glutamate buffered G-CSF composition provided in step (a) hasa volume of from 100 ml to 4 l.
 7. The method of claim 1, wherein theG-CSF composition in step (b) is cooled to a temperature of between −15°C. and −25° C. or a temperature of between −60° C. and −80° C.
 8. Themethod of claim 1, wherein the frozen G-CSF composition in step (c) isstored over a period of at least three months.
 9. The method of claim 1,wherein temperature increase of the frozen G-CSF composition to atemperature within the range of from 2° C. to 8° C. in step (d) iseffected by gradually or linearly increasing temperature over a periodof time of at least 6 hours.
 10. The method of claim 1, whereintemperature increase of the frozen G-CSF composition to a temperature instep (d) is effected by transferring the frozen composition to atemperature within the range of from 2° C. to 8° C. and maintaining thecomposition at said temperature for a period of time of at least 12hours.
 11. The method of claim 1, wherein the G-CSF composition is freeof surfactant.
 12. The method of claim 1, wherein the G-CSF compositioncomprises a surfactant.
 13. A method of providing a pharmaceuticalcomposition of non-glycosylated recombinant human G-CSF, said methodcomprising the steps of: (a) formulating non-glycosylated recombinanthuman G-CSF with an acetate or glutamate buffer and sorbitol to obtainan aqueous buffered G-CSF composition, wherein the amount of G-CSF is inthe range of from 0.1 mg/ml to 8.0 mg/ml and wherein said compositionhas a volume of from 100 ml to 8 l; (b) cooling the G-CSF composition ofstep (a) to a temperature of −15° C. or below to obtain a frozen G-CSFcomposition; (c) storing the G-CSF composition obtained in step (b) inthe frozen state over a period of at least one month; (d) increasing thetemperature of the frozen G-CSF composition of step (c) to a temperaturewithin the range of from 2° C. to 8° C. over a period of time of atleast 6 hours to allow the composition to thaw and to obtain a liquidcomposition having a G-CSF content of at least 95% of the G-CSF contentof the composition provided in step (a); and (e) filling the liquidcomposition obtained in step (d) into primary packagings for parenteraluse.